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Biological nitrification inhibition (BNI) in the tropical grass <i>Brachiaria humidicola</i> could reduce net nitrification rates and nitrous oxide (N₂O) emissions in soil. To determine the effect on gross nitrogen (N) transformation processes and N₂O emissions, an incubation experiment was carried out using ¹⁵N tracing of soil samples collected following 2 years of cultivation with high-BNI <i>Brachiaria</i> and native non-BNI grass <i>Eremochloa ophiuroide</i>. <i>Brachiaria</i> enhanced the soil ammonium (NH₄⁺) supply by increasing gross mineralization of recalcitrant organic N and the net release of soil-adsorbed NH₄⁺, while reducing the NH₄⁺ immobilization rate. Compared with <i>Eremochloa</i>, <i>Brachiaria</i> decreased soil gross nitrification by 37.5% and N₂O production via autotrophic nitrification by 14.7%. In contrast, <i>Brachiaria</i> cultivation significantly increased soil N₂O emissions from 90.42 μg N₂O-N kg⁻¹ under <i>Eremochloa</i> cultivation to 144.31 μg N₂O-N kg⁻¹ during the 16-day incubation (<i>p</i> < 0.05). This was primarily due to a 59.6% increase in N₂O production during denitrification via enhanced soil organic C, notably labile organic C, which exceeded the mitigated N₂O production rate during nitrification. The contribution of denitrification to emitted N₂O also increased from 9.7% under <i>Eremochloa</i> cultivation to 47.1% in the <i>Brachiaria</i> soil. These findings confirmed that <i>Brachiaria</i> reduces soil gross nitrification and N₂O production via autotrophic nitrification while efficiently stimulating denitrification, thereby increasing soil N₂O emissions.